- Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long‐chain polyunsaturated fatty acid (LC‐PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic‐resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched‐chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild‐type and mutant proteins were characterized in the AHAS‐deficient E. coli, BUM1 strain. Cloned PiAHAS wild‐type and mutant genes complemented AHAS‐deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild‐type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type. Biotechnol. Bioeng. 2012;109: 2340–2348. © 2012 Wiley Periodicals, Inc.